The present invention provides compositions and methods for identification of F-box proteins, as well as for drug discovery and assessment. In particular, the present invention provides components of an E3 complex involved in ubiquitination of cell cycle regulators and other proteins, as well as members of a class of proteins that directly function in recognition of ubiquitination targets.
The proper development and maintenance of a multicellular organism is a complex process that requires precise spatial and temporal control of cell proliferation. Cell proliferation is controlled via an intricate network of extracellular and intracellular signaling pathways that process growth regulatory signals. This signaling network is superimposed upon the basic cell cycle regulatory machinery that controls particular cell cycle transitions. In eukaryotes, the cell cycle is comprised of an ordered series of discrete events. In contrast to the periodicity of eukaryotic DNA replication and mitosis, cellular growth requires that most metabolic reactions occur continuously. The cell cycle regulatory machinery coordinates the events that occur during the cell cycle, as well as cell growth. Protein degradation is an important aspect of the development and maintenance of multicellular organisms, as it provides direction, order, and the appropriate timing for the key events that occur during the cell cycle.
The problem of how cell division is controlled has long been a topic of intense research. Early models suggested the existence of an initiator that would accumulate during the cell cycle, and induce DNA replication or mitosis when it reached a critical concentration. The mitotic process would then inactivate the initiator, thereby xe2x80x9cresettingxe2x80x9d the cell cycle. Subsequent research showed that mitotic cyclins accumulate during interphase to drive entry of cells into mitosis. These cyclins are then degraded at the end of mitosis, in order to reset the cycle. Protein degradation has been shown to have a pervasive role in the regulation of cell cycle progression. For example, proteolysis is required for multiple mitotic processes, and for initiating DNA replication (See, King et aL, Science 274:1652-1659 [1996]). Nonetheless, much remains unknown regarding the proteins and the interactions that are involved in the proteolytic regulation of the cell cycle and other processes. Indeed, many proteins are likely to be involved in proteolysis and cellular maintenance (as well as other processes). Such information is needed for the development of compounds to regulate the cell cycle and prevent or treat diseases associated with abnormal cell proliferation.
The present invention provides compositions and methods for gene identification (e.g., F-box genes), as well as drug discovery and assessment. The present invention provides components of an E3 complex involved in ubiquitination of cell cycle regulators and other proteins, as well as members of a class of proteins that directly function in recognition of ubiquitination targets.
Thus, the present invention provides the function of a class of proteins referred to as F-box proteins in targeted ubiquitination. The present invention finds utility in methods for developing compounds that affect ubiquitination. The present invention also provides numerous novel F-box containing mammalian genes whose encoded proteins are contemplated to function in processes including, but not limited, to targeted ubiquitination of cellular proteins.
The present invention also provides amino acid and DNA sequence information for eighteen novel F-box-containing human or mouse genes. As with Cdc4, Grr1, Skp2, and cyclin F, these novel F-box proteins have the capacity to associate with Skp1 and to simultaneously interact with other proteins through other protein-protein interaction motifs encoded by regions of their genes other than the F-box. Thus, the present invention provides compositions and methods for determining the interaction of these proteins with other proteins.
In one embodiment, the present invention provides an isolated polypeptide comprising at least one functionally active fragment of an F-box protein. In a preferred alternative embodiment, the F-box protein is mammalian, while in a particularly preferred embodiment, the F-box protein is human or murine.
In another embodiment, the functionally active fragment comprises the amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOS:1, 3, 5, 9, 13, 17, 19, 25, 27, 41, 45, 47, 51, 53, 55, and 57, while in alternative embodiment, the functionally active fragment comprises the amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOS:7, 11, 15, 21, 23, 29, 31, 33, 35, 37, 39, 43, and 49.
The present invention also provides a purified antibody which binds specifically to the isolated polypeptide encoding an F-box protein. In one embodiment, the antibody is monoclonal, while in another embodiment, the antibody is polyclonal. In another embodiment, the present invention provides a purified antibody which specifically binds to a complex comprised of an F-box protein and an F-box protein target. In yet another embodiment, the present invention provides an antibody which specifically binds to a complex comprised of an F-box protein and Skp1; it is contemplated that the Skp1 in the complex may be bound to another protein, but such binding is not required.
The present invention also provides an isolated nucleotide sequence encoding at least one functionally active fragment of an F-box protein, wherein the nucleotide sequence encodes at least a portion of an F-box protein. In a preferred embodiment, the F-box protein is mammalian, while in particularly preferred embodiments, the F-box protein is human or murine. In one embodiment, the isolated nucleotide sequence comprises at least a portion of the sequence set forth in SEQ ID NOS:2, 4, 6, 10, 14, 18, 20, 26, 28, 42, 48, 52, 54, 56, and 58. In another embodiment, the isolated nucleotide sequence comprises at least a portion of the sequence set forth in SEQ ID NO:8, 12, 16, 22, 24, 30, 32, 34, 36, 38, 40, 44, and 50.
The present invention also provides a vector comprising a nucleotide sequence, wherein the nucleotide sequence comprises the nucleotide sequence encoding at least one functionally active fragment of an F-box protein, wherein the nucleotide sequence encodes at least a portion of an F-box protein. In one preferred embodiment, the isolated nucleotide sequence comprises at least a portion of the sequence set forth in SEQ ID NOS:2, 4, 6, 10, 14, 18, 20, 26, 28, 42, 48, 52, 54, 56, and 58, while in another preferred embodiment, the isolated nucleotide sequence comprises at least a portion of the sequence set forth in SEQ ID NO:8, 12, 16, 22, 24, 30, 32, 34, 36, 38, 40, 44, and 50.
The present invention also provides a host cell transformed with at least one vector comprising a nucleotide sequence, wherein the nucleotide sequence comprises the nucleotide sequence encoding at least one functionally active fragment of an F-box protein, wherein the nucleotide sequence encodes at least a portion of an F-box protein. In one preferred embodiment, the isolated nucleotide sequence comprises at least a portion of the sequence set forth in SEQ ID NOS:2, 4, 6, 10, 14, 18, 20, 26, 28, 42, 48, 52, 54, 56, and 58, while in another preferred embodiment, the isolated nucleotide sequence comprises at least a portion of the sequence set forth in SEQ ID NO:8, 12, 16, 22, 24, 30, 32, 34, 36, 38, 40, 44, and 50.
The present invention also provides an isolated nucleotide sequence encoding the amino acid sequence selected from group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57. In one embodiment, the present invention provides a vector comprising an isolated nucleotide sequence encoding the amino acid sequence selected from group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15 , 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57. In another embodiment, the present invention provides a host cell transformed with this vector.
The present invention further provides a polynucleotide sequence comprising at least fifteen nucleotides, which hybridizes under stringent conditions to at least a portion of a polynucleotide sequence, wherein the polynucleotide sequence is selected from the polynucleotide sequences set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, and 58.
The present invention also provides methods for detection of polynucleotides encoding F-box protein in a biological sample comprising the steps of: hybridizing at least a portion of the polynucleotide encoding an F-box protein, to nucleic acid material of a biological sample, thereby forming a hybridization complex; and detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding F-box protein in the biological sample. In one embodiment of the method, prior to hybridization, the nucleic acid material of the biological sample is amplified by the polymerase chain reaction.
The present invention also provides methods for the detection of F-box protein targets comprising the steps of: providing an F-box protein, and a sample suspected of containing an F-box protein target; exposing the F-box protein to the sample under conditions such that the F-box protein binds to the F-box protein target to form an F-box protein and target complex; and detecting the F-box protein and target complex. In one embodiment of the method, the box protein target is selected from the group consisting of cyclins, cyclin-dependent kinases, and IxcexaB. An alternative embodiment further comprises the step of analyzing siad F-box protein and target complex, wherein the analyzing comprises observing the F-box protein and target complex for degradation of the F-box protein target. In another embodiment, the method further comprises the step of exposing the F-box protein and F-box protein target to an F-box protein antagonist. In yet another embodiment of the method, the F-box protein antagonist prevents the formation of the F-box protein and the target complex.
The present invention also provides methods for the detection of an F-box protein and Skp1 complex, comprising the steps of: providing an F-box protein, and Skp1; exposing the F-box protein to Skp1 under conditions such that the F-box protein binds to Skp1 to form an F-box protein and Skp1 complex; and detecting the F-box protein and Skp1 complex. One embodiment of the method further comprises the step of exposing the F-box protein and Skp1 to an F-box protein antagonist. In yet another embodiment of the method, the F-box protein antagonist prevents the formation of the F-box protein and Skp1 complex.
The present invention also provides methods and compositions useful to determine the complexity and diversity of mammalian F-box proteins, as well as the identity of F-box proteins from various species, the protein-protein interaction domains involved, the proteolytic pathways, and regulatory pathways. Indeed, the present invention provides methods and compositions to identify the functions and ubiquitination targets of these and other F-box containing proteins.
However, the present invention is not limited to F-box proteins involved in ubiquitination. Thus, the function of F-box proteins is not necessarily limited to ubiquitination, and the present invention provides the methods and compositions to make this determination. It is contemplated that additional F-box containing genes will be discovered through the use of two-hybrid screens with Skp1 or ubiquitination targets as the two-hybrid xe2x80x9cbaitxe2x80x9d (e.g., as described in the Example 6). It is also contemplated that additional F-box genes will be discovered through sequencing of the mammalian genome and sequence analysis, to determine the homology with existing F-box proteins, such as those identified in the present invention.
The present invention also provide compositions and methods for development of drugs that disrupt at least one pathway in which F-box proteins function, and are required for biological and/or biochemical processes.
The present invention also provides methods and compositions to identify and/or investigate cell cycle regulators, transcription regulators, proteins involved in DNA replication, and other cellular regulatory proteins. It is further contemplated that the present invention finds use in elucidating inflammatory response and infectious disease processes involving protein degradation, as well as development of compounds that control (i.e., either enhance or retard) protein degradation, as appropriate to ameliorate the effects of the inflammatory response or disease process.
The present invention also provides methods and compositions for identifying and investigating the function of protein targets whose abundance is altered in disease, as well as for detection, identification, and characterization of mutations in F-box genes through various methods, including, but not limited sequence analysis, Southern blot analysis of DNA, etc. Furthermore, the present invention also finds use in assessing alterations in cellular protein abundance due to overexpression of particular F-box proteins. It is contemplated that such alterations are associated with particular diseases. The present invention also finds use in determination of overexpression caused by gene amplification in DNA samples from diseased tissue or individuals through such methods as Southern analysis using a particular F-box gene as probe.
It is also contemplated that targets of novel human F-box proteins will be determined by those experienced in the art by approaches including, but not limited to two-hybrid library screens, immunoprecipitation analysis followed by immunoblotting with antibodies against candidate targets, peptide mapping, mass spectral analysis, peptide sequencing, and/or by screening lambda based expression libraries with F-box protein probes.
In addition, the present invention finds use in engineering F-box proteins to artificially recruit particular proteins into an E3 complex for ubiquitination. Thus, it is clear that the present invention provides methods and compositions for detailed investigation of F-box proteins, as well as proteins that associate with F-box proteins. Furthermore, the present invention thereby provides methods and compositions for the detection and analysis of abnormalities in proteolytic functions, as well as methods and compositions for the development of compounds suitable for use in ameliorating such abnormalities.